Article pubs.acs.org/JPCB
Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain Agnieszka A. Gil,† Sergey P. Laptenok,‡ Jarrod B. French,† James N. Iuliano,† Andras Lukacs,‡,# Christopher R. Hall,‡ Igor V. Sazanovich,§ Gregory M. Greetham,§ Adelbert Bacher,∥ Boris Illarionov,⊥ Markus Fischer,⊥ Peter J. Tonge,*,† and Stephen R. Meech*,‡ †
Department of Chemistry, Stony Brook University, New York 11794-3400, United States School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, U.K. § Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, Oxon OX11 0QX, U.K. ∥ Department Chemie, Technische Universität München, D-85747 Garching, Germany ⊥ Institut für Biochemie und Lebensmittelchemie, Universität Hamburg, Grindelallee 117, D-20146 Hamburg, Germany # Department of Biophysics, Medical School, University of Pecs, Szigeti ut 12, 7624 Pecs, Hungary ‡
S Supporting Information *
ABSTRACT: The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the β-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.
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INTRODUCTION
The LOV domain is a member of the Per-Arnt-Sim (PAS) superfamily, specifically of a subfamily which binds a flavin molecule in a binding pocket well protected from the surrounding medium (Figure 1). The isoalloxazine unit of the flavin mononucleotide cofactor (FMN, see Supporting Information Figure S1 for the structure) acts as the chromophore, absorbing light at about 450 nm in the unilluminated (dark) state. Following singlet state excitation, the flavin triplet state is formed and reacts (via a mechanism which has yet to be fully characterized) with a conserved cysteine residue, generating a covalent bond between the cysteine sulfur and the C4a atom of the isoalloxazine chromophore (Figure S1 for atom numbering). This cysteinyl adduct absorbs at 390 nm (hence designated A390) and
The LOV (light-oxygen-voltage) domain is a versatile blue light sensing flavoprotein motif found in plants, fungi, and bacteria. This modular unit is coupled to a diverse range of signaling output domains and thus involved in the optical control of a variety of functions, including the phototropic response, circadian rhythms, and gene expression.1−5 This combination of diversity and modular nature led to the adoption of the LOV domain as a key element in optogenetics, where light induced structure change has been recruited to optically regulate a range of activities, such as the tryptophan repressor,6 dihydrofolate reductase,7 and GTPase RAC1,8 among others.9,10 Consequently, there is wide interest in establishing a detailed microscopic picture of its light sensing mechanism, from both a fundamental point of view and to facilitate the rational engineering of new optogenetic applications. © 2017 American Chemical Society
Received: January 4, 2017 Published: January 9, 2017 1010
DOI: 10.1021/acs.jpcb.7b00088 J. Phys. Chem. B 2017, 121, 1010−1019
Article
The Journal of Physical Chemistry B
be visualized in one experiment with very high signal-to-noise. Interrogation of TRMPS data using isotope-labeling and sitedirected mutagenesis then enables the mechanism of photoactivation to be delineated in unprecedented detail.
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EXPERIMENTAL SECTION Time-Resolved Multiple Probe Spectroscopy (TRMPS) Measurements. The femtosecond to millisecond timeresolved infrared (TRIR) data were recorded using the timeresolved multiple probe spectroscopy (TRMPS) apparatus recently developed at the Central Laser Facility. This has been described in detail elsewhere22 but essentially allows the measurement of high signal-to-noise (500
a Accuracy of all time constants better than ±5%. bResults from a parallel fit to five isotopologues. cSecond component not detected. d Second fast component required to fit the data, suggesting departure from exponential relaxation at early time.
that 3FMN is formed from 1FMN* in 2.4 ns. The 3FMN then relaxes in an apparent two-step fashion with time constants of 9.5 and 17 μs to form A390 (the quality of fit data showing the requirement for a second component are included in Figure S2). The evolution associated spectra (EAS, see methods) at 9.5 μs contains vibrational modes characteristic of both 3FMN and A390. This requirement for an additional component between 3FMN and A390 does not therefore indicate resolution 1015
DOI: 10.1021/acs.jpcb.7b00088 J. Phys. Chem. B 2017, 121, 1010−1019
Article
The Journal of Physical Chemistry B
conjugation, consistent with the observed blue shift in electronic spectra.13 The protein bleach at 1690 cm−1 observed in the first EAS is unshifted by formation of A390, while that at 1670 cm−1 is now obscured by a new transient at 1665 cm−1. This transient is present in [apoprotein-13C]-AsLOV2, suggesting it arises from A390. Significantly, in the same EAS, a new transient appears at 1638 cm−1 with concomitant bleaches at 1625 and 1652 cm−1. All three are uniformly shifted down in frequency in [apoprotein-13C]-AsLOV2 (Figure 4B), suggesting they arise from changes in the protein structure induced as a result of A390 formation. Thus, the onset of A390 formation already has a large effect on the protein vibrational spectrum. The second phase of A390 formation is characterized by a 17 μs time constant and reveals further evolution in the distribution of the higher wavenumber protein modes, most notably a strongly increasing bleach at 1625 cm−1. These kinetics show that evolution in the protein structure occurs on the microsecond time scale, while the wavenumber (1625 cm−1) suggests assignment to a structural perturbation of a βsheet amide I mode (which are observed in the 1615−1645 cm−1 range in D2O43); this is consistent with the simulations of Peter et al., who report that A390 formation leads to reorganization of Q513 which transmits stress to adjacent βstrands.42 These changes in protein structure are accompanied by formation of a new transient at 1540 cm−1 near the intense 1550 cm−1 isoalloxazine bleach; this transient is of mixed character, showing a small shape change on [apoprotein-13C] substitution, while the TRIR of three samples with 13C labeled flavin cofactors (Figure 4C−E) confirms it is associated with A390 formation. This is consistent with changes in protein structure and A390 formation occurring simultaneously, reflecting dispersive kinetics. The comparison of TRIR and FTIR data in Figure 2B pointed to continued spectral evolution on a longer (hundreds of microseconds) time scale. Such a long time constant cannot be determined accurately using the kHz repetition rate apparatus, because the sample must be rastered and flowed (see online methods) and thus necessarily replaced on this slow time scale. However, such sample replacement will affect all modes in the same way, so by normalizing TRIR spectra to a characteristic chromophore mode which remains constant after A390 formation, the slow evolution can be visualized. The result is shown in Figure 5 where data beyond 100 μs were normalized to the adduct 1552 cm−1 bleach. There is clearly continuing evolution in protein amino acid modes on the hundreds of microsecond time scale, while the 1722 cm−1 A390 isoalloxazine C4O mode remains, as expected, unaltered. The largest change is at 1640 cm−1, where relaxation at 100 μs was incomplete (Figure 2B)this probably reflects relaxation in an α-helix backbone (typically around 1650 cm−1 43). There is also a smaller but significant evolution at 1672 cm−1. Interestingly, the strong bleach seen in the steady state FTIR difference spectrum at 1645 cm−1 (Figure 2B) is not recovered within 1 ms, which points to ongoing structural relaxation on longer time scales, consistent with the observations of Pfeiffer et al.26 The observations of a series of ever slower kinetics following decay of 3FMN is characteristic of hierarchical relaxation dynamics in the protein structure stimulated by A390 formation. Mutants of AsLOV2. C450 is essential for adduct formation through its reaction with 3FMN,2,13 while Q513 reorganization is believed critical in initiating the broader
parts A and B of Figure 3, revealing a key role for protein modes in the TRIR data, while parts C−E of Figure 3 allow better discrimination of chromophore modes from modes of the perturbed protein backbone. However, these comparisons are best made on the basis of the EAS (Figure 4). EAS from the global kinetic analysis of the complete TRIR data for wild type AsLOV2 are shown in Figure 4A, while in Figure 4B−E the wild type EAS are compared with the four isotopically labeled samples. The comparison with [apoprotein-13C]-AsLOV2 (Figure 4B) resolves contributions to the TRIR spectra of vibrational modes associated with the chromophore from those due to surrounding amino acid residues perturbed by electronic excitation. The first EAS is associated with 1FMN/1FMN*. The major changes induced by 13 C exchange occur above 1600 cm−1, which confirms the contribution of amino acid modes in that region, in addition to the expected two FMN CO mode contributions.29 In particular, the 1690 and 1670 cm−1 bleaches are downshifted in [apoprotein-13C]-AsLOV2 to merge with and contribute to the intense 1645(−)/1618(+) pair, which overlaps the 1615 cm−1 isoalloxazine transient absorption. Further, these modes are present in Figure 4C and D, so they must therefore be of amino acid rather than isoalloxazine origin. In contrast, modes below 1600 cm−1 are unshifted in the first EAS in Figure 4B and barely shifted in Figure 4C and D, demonstrating that they are dominated by isoalloxazine vibrations; the larger perturbation of these transitions at lower wavenumber in Figure 4E suggests that they arise from ring modes of isoalloxazine. The peak at 1705 cm−1 which is unshifted in Figure 4B and C is the C4O localized isoalloxazine mode (confirmed by the shift in [4,10a-13C2]-AsLOV2, Figure 4D). The downshift of the 1670 cm −1 protein mode suggests that the expected isoalloxazine C2O mode (see Figure 2A) in AsLOV2 must have been obscured by it; as a check, polarization resolved experiments were performed which indeed reveal the wild type 1670 cm−1 mode to be composite (Figure S4). Thus, we assign the shoulder seen at 1660 cm−1 in wild type, and revealed unshifted in the [apoprotein-13C]-AsLOV2 EAS, to the C2O localized isoalloxazine mode which is supported by the [2-13C]AsLOV2 data, Figure 3C. The major contributions from perturbed amino acid modes occur at wavenumber >1660 cm−1, which is too high to be assigned to amide I vibrations, so the most plausible assignment for the 1690 and 1670 cm−1 bleaches is to carbonyl containing side chains which interact with isoalloxazine. The X-ray structure of AsLOV2 (Figure 1) suggests a prime candidate to be the conserved glutamine residue, Q513 (Figure 1), which had already been proposed to be involved in triggering protein structure change.19,42 The second EAS reflects formation of 3FMN. The main changes are associated with isoalloxazine transient absorptions, with negligible changes in modes >1600 cm−1; the amino acid modes perturbed on electronic excitation are unaffected by intersystem crossing (Figures 3 and 4). The first phase of the reaction between 3FMN and the adjacent C450 to form A390 is characterized by spectral evolution from the second to third EAS. The latter comprises both triplet modes (1438, 1488 cm−1) and modes associated with formation of the final A390 state. As discussed above, this mixed character is assigned to a spectroscopic manifestation of underlying dispersive kinetics. A transient at 1722 cm−1 unshifted in [apoprotein-13C]-AsLOV2 (Figure 4B) but absent in Figure 4E is assigned to the C4O mode of A390; the increase in frequency results from a loss of 1016
DOI: 10.1021/acs.jpcb.7b00088 J. Phys. Chem. B 2017, 121, 1010−1019
Article
The Journal of Physical Chemistry B
effect on the TRIR spectra to Q513A, but, surprisingly, quite different 3FMN to A390 kinetics, with the reaction in Q513L being almost twice as fast as in Q513A (Figure S6). We propose that changes in steric interactions with the larger nonpolar leucine side chain caused cysteine 450 to move closer to the isoalloxazine ring; covalent bond formation (whatever the mechanism) is expected to be very distance dependent. Most significantly, in Q513A, the 1640 cm−1 protein mode in A390 has developed to a much lesser extent than in wild type AsLOV2 within the first 100 μs (Figure 6). Further, the slower structural evolution seen in AsLOV2 at 1640 cm−1 (Figure 5) and assigned to changes in α-helix amide I modes is absent in Q513A (Figure S7). The Q513A mutation causes major changes in the first EAS (Figure 6). As noted by Nozaki et al.,19 the FMN C4O bleach shifts up from 1705 to 1720 cm−1, indicating a weaker H-bonding interaction between FMN carbonyl and adjacent amino acid residues, consistent with the structure (Figure 1) which shows C4O H-bonded to Q513 and N482. Significantly, both perturbed amino acid modes identified at 1690 and 1670 cm−1 are absent in Q513A, proving that their origin lies in the strong coupling between the isoalloxazine ring and the H-bonding network, mediated by Q513. We propose that perturbation of the H-bonding environment around the isoalloxazine ring on electronic excitation arises from a change in electron density between S0 and S1. Such a change would modify H-bond strengths, giving rise to the instantaneous perturbation observed (Figures 2 and 6). We note that a similar instantaneous change was observed in a blue light using flavin (BLUF) domain protein, in which glutamine reorganization is also proposed to be a key step in signaling state formation.37 In that case, the effect of electronic excitation has been discussed in terms of light induced structure change in the glutamine.45−50 Evidence that changes in isoalloxazine electronic structure can lead to optically induced changes in its interaction with its protein environment have also been reported in the calculations and Stark shift measurements of Kodali et al.51 A similar mechanism could operate in AsLOV2, with electronic excitation modifying the H-bond environment prior to A390 formation.
Figure 5. Long time apoprotein structural evolution. Evolution of the 1540 cm−1 normalized TRIR spectra measured for hundreds of microseconds after adduct formation, showing continued evolution in the protein structure. Kinetics at key wavenumbers are shown as an inset. Irregular spacing of data points in the inset reflects collection of more data at early time and the multiplex nature of the TRMPS experiment.22
structure change in the protein.19,44 Mutation at these residues thus probes the AsLOV2 photocycle mechanism. The most significant EAS recovered from the analysis are shown in Figure 6 and in more detail in Figure S5.
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CONCLUSION Structural evolution in AsLOV2 from 0.1 ps to 1 ms after absorption of a photon has been probed using high signal-tonoise IR difference spectroscopy to study the protein, its isotopologues, and two mutants. These data point to a detailed mechanism for signaling state formation in AsLOV2, summarized in Figure 7. Electronic excitation leads directly (
